Construction In Vitro Of Composited Tissue-engineered Skin Using Human Keratinocyte Stem Cells And Dermal Fibroblasts

In 1987, the word of tissue engineering was fisrt put forword in Wastington science fund committee. It is recognized as one of the main hotspots in the 21st century after molecular biology and cell biology. Tissue engineering is an emerging multidisciplinary frontier science involving molecular biology, cell biology, bioengineering, and clinical medicine that is likely to revolutionize the ways to improve the health and quality of life for millions of people worldwide by restoring, maintaining or enhancing tissue and organ function. The emerging field of tissue engineering focuses on the creation of living tissues and organs for use in tissue repair and transplantation. Tissue engineered skin is the best technique in tissue engineering and constructed by cultured seed cells in vitro provides a new way to accelerate skin wound healing and improve the quality of tissue repair. This review article focuses upon the development of skin seed cells, dermal scaffold materials, their construction and application. Human cells are seeded onto biocompatible scaffolds and grown under physiologic conditions to produce all-human biointeractive implants. Tissue-engineered skin implants have shown efficacy in a variety of wound applications.The study of seed cells is the base and upstream. Stem cells possess self-renewal, long-term cell viability, and differentiation potential, which make them the best seed cells. To avoid possible ethical problems and immunologic rejection about embryonic stem cells, adult stem cells are considered a more logical population and gently become the hotspot for tissue engineering. Keratinocyte stem cells are adult stem cells derived from ectoderm, possessing potent proliferative capacity and self-renewal and differentiation potential capacity. They are considered the key resource for epidermal and skin appendage regeneration. But the number of keratinocyte stem cells is low and only occupy 1~10% of basal layer cells in the epidermis, therefore the key problem is to enrich sulficient keratinocyte stem cells. Fibroblasts are the main cells in dermis. They not only have the ability of division and proliferation but also synthosis and excrete dermal matrix protein. They can promote the proliferation, moving and maturity of keratinocytes, and likely to be the best seed cells in tissue engineering. The significant roles of scaffold materials in tissue engineering are to offer the space of cell proliferation and to lead tissue reconstitution. The collagen gel from rat tail has been often used as the extracellular matrix in cell culture in vitro. It shows low immunogenicity and good cytocompatibilityWe have obtained high homogenous human keratinocyte stem cells and dermal fibroblasts by the co-enzyme digestion method, rapid adhesion method and serum-free culture system and prepared dermal scaffold using the collagen from rat tails. The composited tissue-engineered skin composed of human keratinocyte stem cells and fibroblasts in collagen-dermal scaffold materials has been developed. The experiments totally can be divided into four parts:1. Isolation and cultivation of human keratinocyte stem cells and identification of their biological characteristics.The epidermis were separated from the dermis intactly using dispaseⅡand were separated into single cell suspensin using trypsin. Cells adherent to type IV collagen-coated dishes within 10~15min were harvested and cultured by defined keratinocytes serum-free medium. The adherent cells were polygord, slabstone appearance, with the ability of colony formation by means of phase contrast microscope. The cells were small, big cell nucleus, in which there were obviously abundant heterochromatin, and rare microvilli and organelles under transmission electron microscopy. The relative specific markers of keratinocyte stem cells, CK15, CK19, p63 and CD29 were positive by immunocytochemistry, immunofluorescent and fluorescence-activated cell sorting. In contrast, vimentin and cytokeratin10, which are the specific markers of fibroblasts and epidermal terminal differeciation respectively, were negative. The cells in G0/G1 stage accounted for 81.475% by cell cycle analysis and suggested slow cell cycling nature. The results of RT-PCR and Westernblot were same to others. The keratinocyte stem cells were induced to differentiate into the epidermal cells of CK10-positive. Thus, a standardized technologic method has been establised for the isolation, cultivation, purification and identification of hKSCs.2. Isolation and cultivation of huam dermal fibroblasts and identification of their biological characteristics.The dermis was separated into single cells using dispaseⅡand colleganase. The cells were cultured with DMED/F12 including 10% fetal calf serum. Until passage 15, the shape of the cells had continued to be fishes and whirlpool-like. Under transmission electron microscopy, the cultured cells many microvilli and organelles,including plenty of enlarged 6 endoplasmic reticulum and chondriosome. The cells were Vimentin-positive with homogeneity of higher than 90% by immunocytochemistry, immunofluorescent and flow cytometry cell sorting. The cultured cells maintained stable biological characteristics and steady proliferation at even passage 15 and stay at the state of un-differentiation in vitro by colony forming efficiency, cell cycle, karyotype analysis, RT-PCR and Westernblot. So, a standardized platform has been established for isolation, cultivation, purification, identification and amplification of human dermal fibroblasts and providing an excellent cell source for tissue engineering.3.Preparation of dermal scaffold materials and chatacteristics of their biological properties.Collagen possesses low immunogenicity and high cytocompatibility. We extracted collagen protein from rat tails by acetic acid with the concentrasion was 0.5mg/ml. The molecular weight was about 80kDa analyzed by SDS-PAGE, and the collagen protein was typeⅠcollagen. Collagen scaffold was prepared by lyophilization and its bio-characteristics were observed by phase contrast microscope, HE staining, scanning electron microscope, laser scan confocal microscope and cell count kit-8 analysis. The scaffolds possessed 3-dimensional structural features and porosity. Its translucency was good. The apertures were uniform and their size was about 200μm. Their water-absorbing capacity was almost 42 times as much as their weight. It showed well void content. To detect the stability in different conditions, the collagen scaffolds were immerged into 75% ethanol, DMEM/F12 and collagenase. The result demonstrated that the scaffold had favorable stability. Human dermal fibroblasts were seeded in the scaffolds to investigate its proliferation and metabolism. Human dermal fibroblasts were well adherent to the surface of the scaffolds and highly connected to each other like a net and grow in a pattern of multilayer by immunocytochemistry, laser scan confocal microscope and so on. Cell count kit-8 analysis showed that the viability and the proliferation state had the characteristic proliferation pattern of fibroblasts in monolayer culture. So, the collagen scaffold showed good cytocompatibility during the cell culture test, where the dermal fibroblasts proliferated very well. To detect the histocompatibility of the scaffolds, it was performed by transplantation in vivo under the skin in the back of Balb/C mice. During the implanted test, the scaffolds showed low immunoreactivity, neovascularization and led the fibroblasts from the mice without obvious tendency to induce inflammation. It appeared to have good biocompatibility and high biological safety. So we created dermal scaffold materials belong to good cytocompatibility, histocompatibility and three dimensional structural features.4.Construction of composited tissue-engineered skin in vitro.The dermal equivalents were composed of human dermal fibroblasts and dermal collagen scaffolds. Tissue-engineered dermis was prepared using human dermal fiblasts before the 15 passage and observed by HE staining, laser scan confocal microscope. After 4d of co-cultivation, the cells infiltrated evenly into the collagen scaffold and proliferated very well. And after 30d of co-cultivation, the extracellular matrix proteins were found in the tissue-engineered dermis. The composited tissue-engineered skin was produced by seeding human keratinocyte stem cells on a dermal equivalents composed of human dermal fibroblasts and collagen scaffolds. Human keratinocyte stem cells seeded on the dermal equivalents formed a stratified and cornified epidermis and expressed typical markers of differentiation, CK10. This model was produced exclusively from human keratinocyte stem cells and dermal fibroblasts and didn't contain any synthetic materials.Collectively, we have successfully isolated, cultured and identified human keratinocyte stem cells and fibroblasts from the skin of adult human. And we have successfully prepared dermal collagen scaffolds appearing the characteristis of low immunoreactivity, good cytocompatibility, histocompatibility and three dimensional structural features without obvious tendency to induce inflammation, so they have good biocompatibility and high biological safety. Using keratinocyte stem cells and dermal fibroblasts from the same adult human and dermal scaffold materials ourselves, the composited tissue-engineered skin has successfully constructed. It is of importance to provide a basis for the clinical engineered repair of the skin defect in case of non-immunological rejection.